CN117417427B - 一种检测内源性自体胰岛素介导的胰岛素抗体的试剂盒 - Google Patents
一种检测内源性自体胰岛素介导的胰岛素抗体的试剂盒 Download PDFInfo
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Abstract
本发明公开了一种检测内源性自体胰岛素介导的胰岛素抗体的试剂盒。该试剂盒包括放射性抗原蛋白CR12C,TBST缓冲液,蛋白A琼脂糖和蛋白G琼脂糖;所述放射性抗原蛋白CR12C是采用S35‑蛋氨酸对抗原蛋白CR12C进行标记后得到。本发明将两段C肽基因序列通过铰链基因连接到R12序列的两侧,构建出相应的质粒,转录翻译后获得的放射性抗原蛋白CR12C可有效的携带放射性信号,且每个蛋白均包含两个C肽抗原结构域,捕获抗体的能力也大为提高;基于以上设计本发明成功建立了放射配体法对CR12C抗体的检测,在后续的竞争性抑制实验中进一步证明了捕获的CR12CA为针对胰岛素的抗体IAA,该IAA为内源性胰岛素介导产生。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种检测内源性自体胰岛素介导的胰岛素抗体的试剂盒。
背景技术
1型糖尿病(T1DM)胰岛自身抗体包括胰岛素自身抗体(IAA),谷氨酸脱羧酶抗体(GAD),酪氨酸磷酸酶抗体(IA2)和锌转运体8自身抗体(ZnT8),是目前T1DM胰岛β细胞自身免疫破坏的最可靠生物标志,对胰岛自身抗体进行检测有助于了解T1DM的自身免疫病程,并对T1DM患者的诊断、鉴别诊断,以及一般人群和患者一级亲属的风险预测有极大的价值。IAA作为最早出现的胰岛自身抗体,在T1DM的鉴别诊断中具有特殊的地位,不仅可以揭示患者的免疫进程,还可能揭示胰岛素用药前后的抗体变化。患者体内的胰岛素来源包括外源性的注射以及内源性的自体产生两种,这两种胰岛素均会导致IAA的产生,但具有完全不同的指示意义。区别这两种IAA对临床诊断有着重要的提示价值,一直以来IAA的检测均使用胰岛素作为抗原来进行抗体的捕获,也无法对这两种IAA抗体进行有效区分,这也是IAA检测存在的难点。
因此,设计能有效检测内源性自体胰岛素(insulin)产生的胰岛素抗体(IAA)的产品具有重要意义。
发明内容
本发明的目的之一是提供一种放射性抗原蛋白CR12C。所述放射性抗原蛋白CR12C是采用S35-蛋氨酸对抗原蛋白CR12C进行标记后得到,所述抗原蛋白CR12C的氨基酸序列如SEQ ID NO.1所示。
在本发明的一个实施例中,将目的基因(两段C肽基因序列通过铰链基因连接到支架蛋白R12基因的两侧)导入载体pTnTTMVector构建得到CR12C质粒,然后通过快速转录翻译质粒,获得放射性标记的抗原蛋白。具体地,质粒结构包括:XhoI(ctcgag)+kozak序列(gccacc)+ATG+CR12C目的序列+终止密码子+XbaI。转录翻译后获得的每个放射性抗原蛋白CR12C包含两个C肽抗原结构域,中间通过铰链蛋白连接(如图1所示),其携带放射性信号的能力得到了有效的翻倍,且两个抗原结构域使捕获抗体的能力也大为提高。
本发明的目的之二是提供一种检测内源性自体胰岛素介导的胰岛素抗体的试剂盒。
所述试剂盒包括:上述放射性抗原蛋白CR12C,TBST缓冲液,蛋白A琼脂糖和蛋白G琼脂糖。
进一步地,所述试剂盒还包括阳性对照和阴性对照。
本发明的目的之三是提供上述试剂盒在制备1型糖尿病诊断试剂中的应用。
本发明将两段C肽基因序列通过铰链基因连接到R12序列的两侧,构建出相应的质粒,转录翻译后获得的放射性抗原蛋白CR12C可有效的携带放射性信号,且每个蛋白均包含两个C肽抗原结构域(如图1所示),捕获抗体的能力也大为提高。基于以上设计本发明成功建立了放射配体法对CR12C抗体(CR12CA)的检测,在后续的竞争性抑制实验中进一步证明了捕获的CR12CA为针对胰岛素的抗体IAA,该IAA为内源性胰岛素介导产生。
附图说明
图1为本发明中放射性抗原蛋白CR12C的模式图。
图2为不同模拟样本添加量下检测的放射读数。
图3为健康人群抗体CR12CA的界值判定结果。
图4为抗体CR12CA在T1DM患者、DM(非T1DM)患者及健康人群中的分布结果。
图5为抗体CR12CA的C肽竞争性抑制实验结果。
图6为抗体CR12CA的insulin竞争性抑制实验结果。
具体实施方式
当前已知胰岛素来源于胰岛素原,胰岛素原同时包含了胰岛素与C肽分子,在随后的加工中被剪切为一个C肽分子和一个胰岛素分子,因此推测早期产生的胰岛素自身抗体(IAA)亦有可能针对的是胰岛素原中的胰岛素分子,此时C肽分子尚未从胰岛素原中剪切下来,也处于被攻击的抗原靶位范围内。因此,发明人假设以C肽分子作为抗原,捕获的既可能是针对C肽的抗体,也有可能是针对包含了部分C肽序列的胰岛素自身抗体,且由于外源型的注射胰岛素是没有C肽结构的,此时捕获的不管是C肽抗体还是胰岛素抗体均是内源性C肽/胰岛素产生的抗体,只需要证实捕获的到底是哪一种抗体即可。
发明人实验室自有S35放射配体法检测平台,通过在质粒的转录翻译过程中添加放射性氨基酸原料S35标记的蛋氨酸,从而获得具有放射性的蛋白质抗原,其原位替换氨基酸的原理不改变任何获得的蛋白质的空间结构,使结果保持很高的特异性,同时其检测范围宽,灵敏性强。该检测平台的应用前提是待翻译的蛋白质序列自身包含一定的蛋氨酸序列,用来进行放射性蛋氨酸的替换,经过前期对C肽的结构评估,发现其完全不含有蛋氨酸,自身无法携带任何放射性信号。发明人通过对基因库上千个基因的筛查,找到了43个不同动物来源的富含蛋氨酸的基因序列,随后对蛋氨酸序列含量进行了计算,排除了25个含量较低的序列;然后对剩下的18个进行质粒合成,并进行了转录翻译,剔除了转录翻译失败以及与人类健康人血清有交叉反应的17个序列,剩下的最后一个基因序列来自加勒比海红鹳(Phoenicopterus ruber),基因名N337_12018,富含蛋氨酸,将其作为支架蛋白命名为R12,运用于该实验。本发明将两段C肽基因序列通过铰链基因连接到R12序列的两侧,构建出相应的质粒,转录翻译后获得的放射性抗原蛋白CR12C可有效的携带放射性信号,且每个蛋白均包含两个C肽抗原结构域(如图1所示),捕获抗体的能力也大为提高。基于以上的改进以及后续的验证实验本发明成功建立了放射配体法对CR12C抗体(CR12CA)的检测,在后续的竞争性抑制实验中进一步证明了捕获的CR12CA为针对胰岛素的抗体IAA,基于以上提及的原理,该IAA为内源性胰岛素介导产生。
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
一、实验材料
1.样本来源:阳性质控样本来自于购买的针对C肽蛋白的商品化抗体。阴性质控血清标本取自于无糖尿病家族史的健康志愿者。糖尿病人(DM)血清978例,其中1型DM(T1DM)722例,非1型DM(非T1DM)256例。健康人379例来自招募人群[年龄(32.3±4.3)岁;男190例,女189例];糖耐量检测(OGTT)空腹及2h血糖正常,排除心、脑、肝、肾等慢性及内分泌疾病,无糖尿病家族病史及自身免疫疾病史。
2.主要试剂及仪器:TNT SP6快速转录翻译试剂盒(L2080,Promega);S35-蛋氨酸(NEG709A 5mCi,PerkinElmer);蛋白A琼脂糖PA(17-5280-02,GE);蛋白G琼脂糖PG(17061805,GE);96孔PVDF平板(3504,Corning);Microscint-20闪烁液(6013621,PerkinElmer);TBST缓冲液(Tris-Base 2.424g,NaCl 8.70g,Tween-20 1.5mL,BSA 1.0g加蒸馏水定容至至1000mL,pH7.4);βCounter液体闪烁计数仪(2450Microplate Counter,Perkin-Elmer);蛋白纯化柱NAP Column(17-0853-02,GE);C肽(035-01,phoenixpharmaceuticals inc);Anti C-Peptide抗体(ab30477polyclonal,abcam);insulin(Novolin@R,novo nordisk);质粒载体pTnTTMVector(L5610,Promega)。
二、实验方法
1.构建CR12C质粒:将两段C肽基因通过铰链基因连接到R12两端,构建出相应的质粒;转录翻译后获得的每个放射性抗原蛋白CR12C包含两个C肽抗原结构域,中间通过铰链蛋白连接,其携带放射性信号的能力得到了有效的翻倍,且两个抗原结构域使捕获抗体的能力也大为提高。
按照哺乳蛋白表达体系进行密码子优化后(避开两个酶切位点)构建在上述质粒载体pTnTTMVector(L5610,Promega)上,包含kozak序列,XhoI、XbaI酶切位点,目的序列。
质粒结构包括:XhoI(ctcgag)+kozak序列(gccacc)+ATG+CR12C目的序列+终止密码子+XbaI。
所述抗原蛋白CR12C其氨基酸序列如下所示:
EAEDLQVGQVELGGGPGAGSLQPLALEGSLQPKPSTPPGSSGGGSIKNILQPGSVDSQTEMVLVNAVYFKGMWEKAFKDEDTQAMPFRMTEQESTPVQMMYQVGSFKVAEMASEKMKILELPYASGELSMLVLLPDDVSGLEEIENAITFEKLTEWTSSSIMEERKIKVYLPRMKMEEKYNLTSVLMALGMTDLFPKPSTPPGSSGGGSEAEDLQVGQVELGGGPGAGSLQPLALEGSLQ(SEQ ID NO.1)。
其中PKPSTPPGSSGGGS为铰链序列。
2.快速转录翻译质粒,获得放射性标记的抗原
S35-蛋氨酸和TNT SP6混合液解冻后置于冰上,依次加入40μL TNT混合液、1μL(1μg/μL)构建的CR12C质粒和5μL S35-蛋氨酸,用4μL无核酸酶水补充至总反应体系为50μL,充分混匀后置于30℃水浴箱孵育90min,随后取出置于冰上准备过NAP-5柱。
取出1只NAP-5柱置于试管架上,将其上下盖子均打开,弃去平衡液后加入1mL的TBST缓冲液平衡NAP-5柱,洗脱3次。将反应混合物小心加在NAP-5柱填充物表面,再用100μL的缓冲液清洗反应管后仍将其加入NAP-5柱中,待红色液体缓慢下移至柱2/3后,加入500μL缓冲液,仔细观察柱下液滴的颜色变化,收集红色过柱液约500μL,从中取出2μL过柱抗原于闪烁管中与1mL闪烁液混匀,置96孔βCounter闪烁计数仪上计数其每分钟脉冲数(countsperminute,CPM)值。
3.待测样本与抗原的结合与检测
每孔各加5μL样本血清或模拟样本,每个标本和质控血清均为双复孔,取适量标记抗原,用6mL TBST缓冲液稀释标记抗原至20000CPM/60μL,向其中每孔加60μL稀释后的标记抗原,每孔CPM值要求≥20000,标记抗原与血清混匀振荡1小时,4℃冰箱中过夜。孵育PVDF板,150μL TBST/孔,4℃冰箱中过夜。次日,倒去PVDF板中的液体,每孔中加25μL的蛋白A/G混合琼脂糖(62.5%的PA与20%的PG按4:1配制),依次从96孔平板各孔中取出50μL混合液转移至96孔PVDF过滤板上,4℃冰箱中混匀1h沉淀抗原-抗体复合物后取出,真空泵抽吸去液体,先向PVDF过滤板各孔中加200μL TBST缓冲液洗涤沉淀物,真空泵抽吸去液体留取沉淀物,再加150μL缓冲液重复洗涤7次,置于烘箱烘干后每孔加入60μL闪烁液,置96孔βCounter计数仪上计数,每孔计数1min。
按下式计算:
放射指数(Index)=(标本血清CPM-阴性质控CPM)/(阳性质控CPM-阴性质控CPM)。
所有数据均采用SPASS26软件进行统计,对所有计量资料符合正态分布的以用均数±标准差表示,采用单因素方差分析、方差分析趋势性检验等。P<0.05为差异显著,具有统计学意义。
三、实验结果
1.放射性抗原蛋白CR12C的有效性判断
转录翻译CR12C质粒,获得带有放射性信号的CR12C抗原,将购买的商业抗C肽抗体作为模拟样本,使用转录翻译的放射性CR12C抗原进行捕获,根据上述实验方法,检测不同模拟样本量下的放射读数。
如图2所示,放射性读数CPM随着模拟样本加样量的递减而递减,经过方差分析趋势性检验P<0.0001,趋势性极显著。因此转录翻译的CR12C抗原具有生物活性,可有效捕获抗C肽抗体,其携带的放射信号随着抗体含量的变化而变化。
后续实验中使用购买的C肽抗体作为阳参,根据信噪比(S/N)>15(S/N:不同C肽抗体添加量的CPM值/阴参的CPM值)以及成本综合考虑,选用0.625μg抗体,CPM约5468.5作为阳参的加样量,如表1。
表1不同C肽抗体添加量下信噪比(S/N)
2.放射配体法检测抗体CR12CA的健康人界值判定
取189例健康人血清进行抗体CR12CA的测定,计算放射指数,取99%百分位点为界值,经计算阳性界值为0.037,阳性判断标准为≥0.037,如图3所示。
3.放射配体法检测抗体CR12CA的批内批间差异
根据抗体CR12CA指数低、中、高在正常人和患者中选择3份血清在批内和批间各重复检测5次(n=5),批内和批间变异系数(coefficient of variance,CV)见表2。结果显示抗体检测指数的批内CV为1.07%~5.06%,批间CV为2.89%~10.56%,阴阳性结果判断重复性100%。
表2抗体CR12CA检测的批内批间差异
4.抗体CR12CA在不同人群中的分布
检测抗体CR12CA分别在T1DM患者、DM(非T1DM)患者及健康人群中的百分比,分别为9.70%(70/722)、4.30%(11/256)及0.53%(1/190),经单因素方差分析P<0.01,差异显著。如图4所示。
5.抗体CR12CA阳性样本的竞争性抑制
取抗体CR12CA阳性的样本进行竞争性抑制实验,在检测中每孔加入2μg购买的商业C肽以及insulin分别进行竞争性抑制实验,结果表明,加入C肽后抗体的放射指数大部份没有明显的变化,未下降至阴性范围;加入insulin蛋白后,抗体的放射指数下降至阴性范围。抗体CR12CA的检测识别的主要是针对insulin的抗体IAA(如图5和图6所示)。
由以上结果可知,本发明的放射性抗原CR12C可有效携带放射性信号,抗体CR12CA检测可有效识别内源性胰岛素产生的IAA,批内批间CV均在有效范围内。此外该实验使用商业抗C肽抗体作为阳参,只是用来对每次实验进行有效的质控,保证实验的系统稳定性,对实验结果及结论并不造成影响。
Claims (3)
1. 一种放射性抗原蛋白CR12C,其特征在于,所述放射性抗原蛋白CR12C是采用S35-蛋氨酸对抗原蛋白CR12C进行标记后得到,所述抗原蛋白CR12C的氨基酸序列如SEQ ID NO.1所示。
2.一种检测内源性自体胰岛素介导的胰岛素抗体的试剂盒,其特征在于,包括:权利要求1所述的放射性抗原蛋白CR12C,TBST缓冲液,蛋白A琼脂糖和蛋白G琼脂糖。
3.根据权利要求2所述的试剂盒,其特征在于,还包括阳性对照和阴性对照。
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